The stapled peptide technology has afforded a novel method for the stabilization of biologically relevant peptide helices. Thus far, it has created unique opportunities for targeting discrete components of complex signaling pathways relevant to the pathogenesis of cancer. The use of this methodology has enabled our study of the apoptotic signaling pathway and, more recently, the manipulation of transcriptional pathways restricted to the nucleus. We aim to significantly evolve the stapled peptide strategy through chemical refinement in order to expand our ability to target pathologic protein interactions implicated in cancer. Throughout the course of our research into the function of p53 family members in cancer (see ZIA BC 011376 project summary), we have found that while our HDM2/HDMX targeting compound SAH-p53-8 restores the transcriptional activity of inhibited p53, several other lines of activity have been found, some of which are entirely independent of p53. In order to provide a chemical approach to target identification, we have developed photactivatable alpha-helices of p53 (pSAH-p53s) which are capable of cross-linking covalently to their target proteins by excitation with UV light. To do this, we have designed our photoactivatable compounds using as a template the sequence of SAH-p53-8, our most active compound to date. Photochemical cross-linking is accomplished by incorporating into the sequence of the peptide a para-benzophenone phenylalanine residue in lieu of an amino acid putatively involved in the requisite protein-protein interaction. As a proof of principle study, we combined a pSAH-p53 with a mixture of its target protein HDM2 and a spectator protein in a superstoichiometric ratio. We found that after exposure to UV light, pSAH-p53 cross-links selectively to HDM2 despite the overwhelming presence of other proteins. With these data on hand, we then proceeded to evaluate the compounds in the complex setting of a cell extract. Gratifyingly, we have found that pSAH-p53s effectively cross-link HDM2 and HDMX from lysates. More importantly, we also found that the pSAH-p53s target other proteins as well. Current efforts are aimed at identifying these novel binding partners and determining their significance to p53 family pathways. In an effort to diversify the use of stapled peptides, we have begun a study using electrochemically-tagged stapled alpha-helices of p53 (eSAH-p53s) to study peptide-protein interactions on an electrode surface in real time. These studies are underway and the progress is reported in the Innovation Award Progress Report section.